Embodiments of this invention were disclosed in Disclosure Documents No. 129,717, dated Aug. 2, 1984, recorded Aug. 6, 1984, and 130094, dated Aug. 14, 1984, recorded Aug. 17, 1984, incorporated by reference, which the Patent and Trademark Office is requested to preserve.
In the past, genetic diseases were diagnosed based on clinical findings once the disease had developed. Various enzyme and protein tests were subsequently developed to confirm or provide more accurate diagnosis and to allow earlier diagnosis. Unfortunately, for many diseases no such tests are available.
Recently, it has become possible to analyze an individual's DNA (which is present in every cell) to determine if certain abnormal genes which will cause genetic diseases are present. These diseases include Huntington chorea, phenylketonuria, thalassemias, and sickel cell anemia. The abnormal genes are found by analyzing restriction site polymorphisms (RSPs) using "Southern blotting" (SB) (Southern, E.M.S., Molecular Biology, 1975, 98:503). This test is time consuming and expensive. However, it is an extremely important method, since it has allowed prenatal diagnosis and thus intervention to prevent birth of severely diseased individuals.
Kan and Dozy, The Lancet 910 (Oct. 28, 1978) described a new approach to prenatal diagnosis of sickel cell anemia utilizing a "Southern blot" of DNA from amniotic fluid cells. When normal DNA was digested with the enzyme Hpa I, the beta-globin gene was contained in a 7.6 kb fragment. In variant DNA, the gene was found in fragments 7.0 kb (hemoglobin-A) or 13.0 kb (hemoglobin-S) in length. The polymorphic Hpa I site detected by this method was not located in the beta-globin gene itself, but rather in an adjacent sequence. Thus "this method of analysis is indirect and suitabe only in those cases where the parents at risk can be shown to carry the appropriate linked polymorphism prior to aminocentesis." Benz, Am. J. Ped. Hematol./Oncol. 6:59 (Spring 1984). (This was done by family studies.).
It is known that sickle cell anemia is caused by a single nucleotide base mutation in the beta globin gene which converts a glutamic acid codon (CAG) to one coding for valine (GTG). Nienhuis, N. Engl. J. Med. 299:195 (1978) proposed direct analysis by means of a restriction enzyme whose recognition site is created or eliminated by the point mutation. His candidate, Mnl I, yield small (60-80 bp) fragments that could not be resolved by blotting techniques at that time.
Wilson, et al., U.S. Pat. No. 4,395,486 (1983) found that direct diagnosis of sickle cell anemia was possible by restriction assay with an enzyme, such as Dde I, recognizing a CTNAG. The B-globin gene fragment was identified by a radiolabeled probe complementary to the 5' end of the gene. Individuals with normal hemoglobin had 175 bp and 201 bp bands; anemic individuals have a single 376 bp band. Unfortunately, the small Dde I generated fragments could be detected and distinguished only after sophisticated technical modification of the blotting techniques.
A new enzyme, Mst I, made possible the use of conventional techniques as directed in Wilson, el al., PNAS (USA) 79:3628 (June 1982). The normal fragment was 1.14 bp long, while sickle cell individuals produced a 1.34 kb fragment.
These fragments are separated according to size by gel electrophoresis. Since many other fragments from the non-globin DNA are also present, a special procedure ("Southern Blot") must be used to find the globin fragments. (Southern, PNAS [USA] 76: 3683-3687 [1979].) After electrophoresis, the DNA is transferred to a filter support (ex., nitrocellulose); then the filter is reacted with a radioactive probe which specifically binds to the globin sequences. This probe will stick to the globin sequences and, following washing and autoradiography, it can be determined whether the patient has 1.34 kb or 1.14 kb length fragments or both. The process of electrophoresis, transfer, filter hybridization, washing, and autoradiography are expensive, time consuming, and, for some size fragments, very difficult.
All of the aforementioned techniques for diagnosing SCA require that the disease create or destroy a restriction site.
Another approach permits one to detect single base changes (point mutations) in genomic DNA even where the change does not alter a restriction site. "Under appropriate hybridization conditions, only perfectly base-paired oligonucleotide-DNA duplexes will form; duplexes containing a single mismatched pair will not be stable." Conner, et al., PNAS (USA) 80:278 (January 1983). In this method, an oligonucleotide complementary to the DNA of a normal (or afflicted) individual in the affected region is synthesized, radiolabeled, and hybridized under stringent conditions. The duplexes are examined by autoradiography. Commenting on this approach, Orkin, BLOOD 63-249 (February 1984) writes: "In order to detect globin DNA fragments or other single-copy sequences in blot hybridization of total DNA, the synthetic probe must be rendered highly radioactive. Our own experience indicates that this is the most troublesome part of the methodology."
Thus, while more versatile than restriction mapping techniques, the stringent hybridization technique shares the disadvantageous requirements for radioactive probes, gel electrophoresis, Southern blotting, filter hybridization, washing and autoradiography. The present invention dispenses with these requirements.
It is known that radiolabelling of probes may be replaced by labeling with biotin, the biotin label then being detected by its affinity with avidin or its binding to an anti-biotin antibody (either then being linked to a reporter enzyme like peroxidase). Renz, EMBO J. 2:817 (1983). The art does not teach, however, use of interactive labels whose interaction is differentially affected by treatment depending on the sequences to which they are bound.
Falkow, U.S. Pat. No. 4,358,935 describes a method of diagnosing an infection utilizing a heterogeneous assay for pathogen DNA or RNA. The assay reagent is a labeled RNA or DNA probe. The patent states that "for the most part" the probe will be radiolabeled. It generaly alludes to the use of labels known in the immunoassay art, but without expression of any preference for a particular nonradioactive label or any discussion of interactive labels. Nor does it mention use of adjacent probes that are differently labeled.
Taber, EP Appl 114,668 discloses a family of DNA probes each of which hybridizes to a different region of a single chromosome of Salmonella bacteria. These probes are preferably radiolabeled, but also may be labeled with biotin. This reacts with avidin to which is bonded a fluorophore, an electron-dense compound, an antibody, or one member of a catalyst/substrate pair. While several probes are preferably used simultaneously, additively increasing the intensity of the resulting signal, there is no suggestion of any interaction among the multiple labels thus associated with the chromosomal DNA. The interactive labels of the present invention afford a much greater increase in sensitivity than that afforded by Taber's noninteractive multiple labels.
Peterson, WO 83/03260 describes a method of determining the polymorphism of human MHC alleles which, like that of Wilson, is dependent on the fractionation of restriction fragments by size. While it recognizes alternative to radioisotopic labels, such as enzymes, enzymatic substrates, co-factors, co-enzymes and luminescent substances, it does not refer to the use of interactive labels.
Ehrlich, EP Appl 84, 796 and Mach, EP Appl 103,960, both relate to HLA (human lymphocyte antigen complex) diagnostic typing based on restriction site polymorphism. As with Wilson, the DNA is restricted and fractionated prior to hybridization. The Mach references refers to alternatives to radiolabeling of hybridization probes, but not to the use of interactive labels.
Ullman, U.S. Pat. No. 3,996,345 teaches an immunoassay system utilizing a fluorescer-quencher pair. In one embodiment, the fluorescer is attached to a first receptor and the quencher to a second receptor for a polyepitopic ligand.
Maggio, U.S. Pat. No. 4,233,402 describes the use of enzyme immunoassay utilizing a reactant label conjugated to an analyte, and a single producing label conjugated to an antibody to said analyte, where the reactant labels acts on a precursor compound to generate a signal mediator which in turn directly or indirectly acts on the signal producing label to generate a signal. Maggio teaches that one label must be attached to the analyte and the other to the receptor. Thus, he teaches against the use of two interactively labeled probes or of a doubly interactively labeled probe. Finally, Maggio does not suggest that interactive label may be used to distinguish a normal DNA sequence from a mutated sequence, let alone suggest a means whereby the difference in sequence operates to affect the interaction of the labels.
Other immunoassay patents of interest are Litman, U.S. Pat. No. 4,275,149; Litman, U.S. Pat. No. 4,299,916; Zuk, U.S. Pat. No. 4,208,479; and Harris, U.S. Pat. No. 4,463,090. The Harris patent deals with cascade amplification of immunoassay signals.
These immunoassays are used to measure trace amounts of organic compounds, not to elucidate fine structure. If the aforestated assays are used to detect a ligand-receptor complex comprising a DNA probe hybridized to sample DNA, they will not be effectual at detecting the lesion. Since stringent hybridization conditions are not used, and the duplex is not cleaved between the labels, the presence or absence of the resulting signal is not dependent on the presence of the lesion. Generally speaking, these immunoassay techniques require special adaptation to detect the fine differences in nucleic acid sequence with which the present invention is concerned.
While the discussion herein focused on prenatal diagnosis of sickel cell anemia, the method of the present invention is equally applicable to other genetic disorders for which the locus of the lesion, and either the normal or mutated sequence about the lesion, are known or isolatable.